A cross-sectional study was conducted to determine the prevalence, risk factors, and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) in 12 beef abattoirs in Gauteng province, South Africa. The relationship between STEC contamination and aerobic bacterial counts on carcasses at various stages of processing was investigated. Multiplex PCR was used to detect virulence genes in broth-enriched samples, to determine O-serogroups in all samples positive for Shiga toxin-encoding genes (stx), and to characterize isolates of STEC. The overall prevalence of STEC determined by PCR in 419 selective enrichment broth samples was 35.1% (147/419), and was significantly higher (P = 0.037) in perineum hide swabs (50%) than in 24 h chilled carcass swabs (20%). The maximum total aerobic plate count (TAPC) was 3.8 log10 CFU/100 cm2 for carcass swabs, but was below the South African microbiological standard for meat export at all stages of carcass processing. There was no significant association between TAPC and STEC contamination. Serogroup O113 was the most prevalent serogroup (13.6%; 20/147) detected. Only 33 isolates, all non-O157 STEC, were recovered, amongst which six different genotype combinations were observed. Additionally, the clinically important serogroups O117, O8, and O2 were isolated. Multivariable logistic regression revealed that the odds of STEC contamination was lower in post-wash (OR = 0.42; 95% CI: 0.18–0.98; P = 0.045) and 24 h chilled (OR = 0.33; 95% CI: 0.12–0.91; P = 0.033) carcass swabs compared to pre- and post-evisceration swabs. It was concluded that non-O157 STEC serogroups more frequently colonize beef cattle slaughtered at abattoirs in our study area than O157 STEC, and therefore have a higher potential to enter the food chain during carcass processing, with food safety implications.