Interspecies relationships within microbial consortia are often associated with mixed microbial infections and are known to impact bacterial resistance to antibiotics. This research examined the interactions among Escherichia, Proteus, and Klebsiella species on antibiotic resistance profiles. Single and mixed cultures of the bacterial isolates were prepared through combinations of two, three and four species. Antibiotic susceptibility testing for mono-cultures was performed by lawn inoculation of Mueller–Hinton agar plates using standardized bacterial suspensions. Gram-negative antibiotic discs were placed on the inoculated plates, which were then incubated at 37 °C for 24 h for mono-culture and mixed-culture plates. Following incubation, zones of inhibition were measured, and compared to evaluate differences in antibiotic susceptibility between single and mixed cultures. E. coli (PX588540) and Kl. pneumoniae (PX588542) exhibited intermediate susceptibility to amoxicillin clavulanate, whereas E. coli (PX588541) and P. mirabilis (PX588543) were resistant. E. coli (PX588541) and P. mirabilis (PX588543) conferred resistance to other members of the mixed culture. In mono-culture assays, both E. coli (PX588540) and E. coli (PX588541) were resistant to cefotaxime; however, an intermediate response was recorded during co-culture. Resistance to gentamicin in mono-culture was observed only in P. mirabilis (PX588543). Kl. pneumoniae (PX588542) was inhibited by nalidixic acid in mono-culture; it demonstrated tolerance to the antibiotic across all co-culture combinations. Additionally, E. coli (PX588541) displayed intermediate susceptibility to nitrofurantoin but became susceptible when co-cultured with E. coli (PX588540). These findings demonstrate that bacterial isolates exhibit different susceptibility and resistance profiles in co-culture compared with pure cultures.