Mycotoxigenic fungi such as Fusarium graminearum, Fusarium verticillioides and Stenocarpella maydis infecting maize grain can be detrimental to both humans and animals due to the toxins they produce. Disease management strategies include tillage practices and crop rotations, however, these have not been sufficiently evaluated in South Africa. The effect of cropping systems on ear rot accumulation and mycotoxin contamination in maize grain was investigated in two localities over a four and six-year period. Cropping systems evaluated were: 1) monoculture maize conventional tillage, 2) monoculture maize no-till, 3) two, and 4) three-year rotation systems consisting of maize/cowpea and maize/cowpea/babala (all no-till), respectively. In Buffelsvallei, two additional crop rotations, maize/sunflower and maize/sunflower/babala (all no-till) were included. Naturally infected trials were visually evaluated for disease severity or incidence while fungal and mycotoxin contamination of maize grain was quantified. Disease incidence and mycotoxin contamination were inconsistent throughout the study period due to seasonal and geographical differences. In Buffelsvallei, cropping system had a significant effect (P < 0.05) on the accumulation of fumonisins and F. graminearum for 2010/11, deoxynivalenol (2011/12) and S. maydis incidence (2013/14). Fusarium graminearum and fumonisin accumulation was significantly higher in the three-year maize/cowpea/babala rotation and two-year sunflower rotation in the 2010/11 season, respectively. Deoxynivalenol levels in monoculture maize, using conventional tillage (2011/12) was significantly higher when compared to all other cropping systems and S. maydis incidence was significantly higher in maize conventionally tilled, no-till and two-year maize/cowpea and maize/sunflower cropping systems in the 2013/14 season. Cropping systems had no significant effects on fungal infection or mycotoxin accumulation in maize grain obtained from trials conducted at Erfdeel. The results of this study indicate that Conservation Agriculture systems under the environments evaluated, did not increase the risk of maize ear rots and mycotoxin production.