This research investigated the variations in the occurrence of Salmonella, STEC O157:H7 and non-O157 in the beef production chain in Colombia affected by seasons, hypothesizing that pathogen prevalence will be highest in the rainy season owing to soil moisture promoting bacteria multiplication and transfer between animals. To test this hypothesis, samples were obtained from five abattoirs, which represent 50% of the beef production in this country. A total of 1017 samples were collected, from which 606 were bovine feces, 206 were hide swabs, and 205 corresponded to carcass post-intervention. From the 1017 samples, 49.9% (n = 507) were collected during dry season, while 50.1% (n = 510) during rainy season. All samples (n = 1017) underwent screening for E. coli O157:H7 and Salmonella, while only a proportion of fecal samples (n = 339) were screened for the big six STEC serogroups and their virulence markers. The effect of season, age of animal and sex of animal were correlated with the prevalence results. A total of 84.7% of fecal samples carried virulence genes associated to STEC (stx or eae), suggesting that testing and control should be increased for the big-six STEC compared to E. coli O157:H7. Pathogen prevalence in feces was found to be 8.3%, 5.0%, and 51.0% for Salmonella, E. coli O157:H7 and STEC non-O157, respectively. Hides had a prevalence of 15.0% and 6.8% of Salmonella and E. coli O157:H7, respectively. Carcasses post-intervention were found to have 4.4% and 2.5% prevalence of Salmonella and E. coli O157:H7, respectively. A seasonal effect was found for fecal samples. E. coli O157 and non-O157 STEC shedding were significantly higher (P ≤ 0.05) during rainy season compared to dry season. In contrast, hides and carcasses were more likely to present lower incidence of pathogens during rainy months compared to dry season; however, it was significant only for Salmonella on carcasses with estimated odds of detection almost six times higher in the dry season relative to the rainy season (OR = 5.90, 95% CI 1.18–29.57).