Clostridium, Salmonella, E. coli and mycotoxin-producing fungi are often involved in opportunistic infection of the GI tract in many livestock species including dairy cattle. However, their abundance in lactating dairy cows with gastrointestinal disease symptoms is poorly understood. The objective of this survey was to quantify common microbial marker and virulence genes in rectal swabs from healthy dairy cows (n = 206) and cows with GI symptoms (n = 159) from commercial farms across 10 US states. The GI symptom group included cows with diarrhea, indigestion, ketosis, displaced abomasum and other digestive issues reported by producers. Genomic DNA was extracted from rectal swabs and tested with a panel of qPCR assays for genus- or species-specific marker or virulence genes from E. coli, Salmonella, Clostridium spp., and Aspergillus. The proportion of positive samples and the quantitative results for each gene in the healthy and GI groups were compared using Fisher’s Exact test and Mann-Whitney tests respectively. Prevalence of stx2, typically associated with Shiga toxin-producing E. coli, the C. perfringens α toxin gene cpa, and the C. difficile toxin B gene tcdB were higher in swabs from sick cows (P < 0.05). Sick cows also harbored higher quantities (P < 0.01) of the E. coli 16S rRNA gene (2.81 vs. 2.27 log₁₀ copies), EAST1, a heat-stable enterotoxin gene in enteroaggregative E. coli, (2.24 vs. 1.82 log₁₀ copies), and cpa (0.74 vs. 0.30 log₁₀ copies). Isolates of E. coli and Clostridium spp. were cultured from swabs and tested in agar cross-streak assays for growth inhibition by several novel Bacillus spp. strains. 813 of 853 E. coli and 390 of 394 C. perfringens isolates were inhibited by at least one Bacillus strain in cross-streak assays. This survey indicates that cows with gastrointestinal symptoms harbor elevated virulence gene quantities from multiple microorganisms, providing new targets for risk assessment, while in vitro inhibition testing shows promise of potential biological solutions for control of opportunistic microbial growth.