Study aim: overtraining (OT) has a detrimental effect on sport performance, but it is not clear to what extent it influences physical fitness. The aim of this study was to examine the relationship between OT and physical fitness in football players.Material and methods: a sample of semi-professional male football players (n = 124) performed a series of anthropometric and physical fitness measurements, and completed the 54-item OT questionnaire of the French Society of Sports Medicine.Results: the OT score was significantly correlated with sit-and-reach test (SAR; rho = −0.20, p < 0.05), theoretical maximal velocity (v0; rho = −0.23, p < 0.05), theoretical maximal force (F0) of the force-velocity test (F-v test; rho = 0.25, p < 0.01) and mean power (Pmean) in W · kg−1 of the Wingate anaerobic test (WAnT; rho = −0.20, p < 0.05). The comparison between OT quartiles revealed that the first quartile scored higher than the third quartile in SAR and in v0 (p < 0.05). The fourth quartile scored higher in F0 than the first, second and third quartiles (p < 0.05). The magnitude of these differences among groups was medium.Conclusions: the negative correlations between OT and physical fitness and the highest scores in fitness for the first OT quartile indicate a negative effect of OT on physical fitness (anaerobic capacity, maximal velocity and flexibility) of football players. In addition, because there is very limited prior relevant research on football players’ OT, our data can be used as reference for future research.