There have not been a single moment in anime or manga where Saitama loses but the moment when Saitama fought Beast King, Boros and Elder Centipede he had to use more than the normal punches that he usually used. In these three the least strongest was Beast King as Saitama used only three normal punches which is not something to hype about.
He just used three Consecutive normal punches which is quite normal as compared to other two. He used those three normal punches to show off in front of the Deep sea King as he was boring Saitama a lot during their fight.
Boros is one of the strongest villain and not the strongest to be introduced. But he made Saitama use more than his normal punches. His armor was quite special as saitama's normal punches did little damage. He only lost his arm as compared to losing his whole body. The other time he caught hold of Saitama's punch very normally and only coughed a small amount of blood.
Saitama used most punches against Boros as he had already used multiple normal consecutive punches and he stood there only due to his ultra regenerative skill. Boros at the other hand cannot be compared with any other as he had the capability to destroy a planet as shown in his fight against Saitama.
So, in order to cope up with Boros, Saitama had to use his Serious punch which ultimately defeats Boros in the end. As the fight ended, Boros's ship fell down on the land. Boros was the first enemy to withstood that much against Saitama.
In the case of Elder Centipede he had to use a Serious punch instead of normal punch which shows that he was not a very easy opponent. It can also be the fact that Elder Centipede could regenerate so in order to stop it from doing so Saitama uses Serious punch in the presence of the king. So, these are the three villains till now against whom Saitama had to use Serious punch.
Who can defeat Saitama?
There is one single guy in anime that Saitama cannot defeat or that can defeat Saitama and that person is Accelerator from To Aru. No power can work against him and if anyone even tries to launch an attack on him he can simply launch it back on him. This thing makes him one of the top overpowered characters in the anime series.
This thing can be accomplished by him simply due to Saitama only being able to use punches or kicks on the other hand Accelerator can use vector and he can apply massive intellect and calculation which normally is impossible. Which he can use to understand any other law which he is not familiar with making it one of the most overpowered ability.
Vector is the ability that is also known as Vector Change and it is the ability which allows him to vector change anything like heat, electricity etc. It have both qualities as it is both passive and active or it can said it is both offensive and defensive.
Due to the passive ability that he have it is impossible for anyone to even lay a finger on him as his auto deflective function will be automatic triggered thus making any kind of attack meaningless on him. For this thing to happen Calculation comes into play as he have to take care of gravity, air pressure, light, oxygen etc to even make this possible.