Background Schistosoma (S.) mansoni infection is endemic in all regions around Lake Victoria and affects all age groups to different degrees. In most endemic areas, less attention has been paid to determining the prevalence of infection, sanitation status, and knowledge about intestinal schistosomiasis (KIS) in fishermen. Therefore, the purpose of this study was to establish the prevalence of S. mansoni infection and associated factors among fishermen in the Busega district. Materials and methods A cross-sectional study was conducted among fishermen in July, 2020 in five fishing villages in the Busega district located along Lake Victoria. A total of 352 fishermen were interviewed with regard to their sanitation status and level of KIS. A single stool sample from fishermen was examined for S.mansoni eggs by using the Formalin-Ether Concentration technique. The potential factors associated with S. mansoni infection were explored using multivariable logistic regression. Results The prevalence of S. mansoni infection was high (65.0%) among fishermen and varied with age, whereby fishermen aged ≤36 years had the highest prevalence. Fishermen had a low level of KIS and the majority of them reported practicing open defecation during fishing (81%). These fishermen with a low level of KIS and who reported defecating in open areas during fishing had 2.8 times (95% CI: 1.0–7.2) and 2.1 times (95% CI: 1.1–3.9) higher odds of being infected with S. mansoni than those with a high level of KIS and those who did not report defecating in open areas during fishing, respectively. Conclusion S. mansoni infection was high among fishermen in the Busega district. Furthermore, fishermen had a low level of KIS and were reported to have defecated in open areas during fishing. Infection with S. mansoni was associated with age, a low level of KIS and open defecation behaviour during fishing. Therefore, mass drug administration (MDA) with praziquantel, health education, and sanitation behaviour change interventions were needed.