This study explores the impact of polystyrene nanoparticles (PS-NPs) and lipopolysaccharides (LPS) on male reproductive health, specifically focusing on testicular function in mice. The research is pioneering in its examination of the combined effects of PS-NPs and LPS. The experiment involved 24 male mice divided into four groups: control, PS-NPs, LPS, and PS-NPs + LPS. Findings revealed that exposure to PS-NPs, LPS, or their combination led to histological alterations in the testes. Notably, the combined exposure to PS-NPs and LPS resulted in a more significant decrease in total sperm count, testosterone levels in plasma and testes, and the expression of steroidogenic acute regulatory (StAR) protein compared to either PS-NPs or LPS alone. Additionally, while both PS-NPs and LPS induced oxidative stress in the testes, the combined exposure did not significantly amplify this effect compared to LPS alone. However, the combination of PS-NPs and LPS did significantly increase the expression of inflammatory factors more than LPS alone, suggesting that PS-NPs exacerbate LPS-induced testicular dysfunction. This study highlights the potential risks to male reproductive health posed by nanoparticles and bacterial infections, contributing valuable insights into the understanding of environmental pollutants’ impact on human health.