Gastrointestinal disease can arise at any time during a dairy cow’s lactation cycle, yet periods of high feed intake and production or greater metabolic demand can increase susceptibility to opportunistic infection. Clostridium, E. coli, Salmonella, and mycotoxin-producing fungi are commonly found in the GI tract, however fluctuation in abundance of these microorganisms or their virulence genes at different stages of lactation remains unclear. The objective of this study was to quantify common microbial marker and virulence genes in rectal swabs from dairy cows during 3 stages of lactation. Rectal swabs were obtained from 370 dairy cows at 26 commercial farms across major US dairy-producing regions, and classified into 3 groups by days post-parturition at time of sampling: fresh (d1–21, n = 95), early lactation (d22–100, n = 146) and late lactation (>101d, n = 129). Genomic DNA was extracted from rectal swabs and genus- or species-specific marker or virulence genes from E. coli, Salmonella, Clostridium, and Aspergillus were quantified using a panel of qPCR assays. Data were log-transformed and the Kruskal-Wallis test, followed by Mann-Whitney pairwise comparisons were performed to detect differences in gene quantity between lactation stage groups. Cows in fresh and early lactation periods harbored higher quantities (H = 7.51, P = 0.02) of EAST1, a heat-stable enterotoxin gene in enteroaggregative E. coli, compared with cows in late lactation (2.06 and 2.13 vs. 1.82 log₁₀ gene copies respectively). Similarly, quantities of Aspergillus 18S rRNA gene (H = 12.93, P < 0.01) and C. difficile toxin A gene tcdA (H = 7.52, P = 0.02) were elevated in fresh and early lactation groups. Fresh cows swabs also contained highest (H = 6.91, P = 0.03) quantities of the C. perfringens α toxin gene cpa compared with cows in early and late periods (0.76 vs. 0.35 and 0.41 log₁₀ copies respectively). These results indicate that harborage of several opportunistic microorganisms and their virulence genes may be greater during the fresh and early lactation periods, providing insight into the increased vulnerability of cows during these stages.