A common herbicide, atrazine, is associated with poor health. Atrazine acts as an endocrine disruptor at supra-environmental levels. Little research, however, has been conducted regarding chronic exposure to environmental atrazine concentrations across generations. This study utilized comprehensive endpoint measures to investigate the effects of chronic exposure to a conservative atrazine concentration (0.02 ng/mL), measured in Australian waterways, on male mice fertility across two generations. Mice were exposed through the maternal line, from the pre-conception period and through the F1 and F2 generations until three or six months of age. Atrazine did not impact sperm function, testicular morphology nor germ cell parameters but did alter the expression of steroidogenic genes in the F1, down-regulating the expression of Cyp17a1 (Cytochrome P450 family 17, subfamily A member 1; p = 0.0008) and Ddx4 (DEAD-box helicase 4; p = 0.007), and up-regulating the expression of Star (Steroidogenic acute regulatory protein; p = 0.017). In the F2, atrazine induced up-regulation in the expression of Star (p = 0.016). The current study demonstrates that chronic exposure to an environmentally relevant atrazine concentration perturbs testicular steroid-associated gene expression that varies across generations. Future studies through the paternal and combined parental lineages should be undertaken to further elucidate the multigenerational effects of atrazine on male fertility.