<p>Polymicrobial infections with <i>Acinetobacter baumannii (A. baumannii)</i> and <i>Candida albicans</i> (<i>C. albicans</i>) are increasingly frequent in urinary tract infections (UTIs). However, experimental data describing their interactions in clinical isolates under co-culture conditions remain limited. In this study, three clinical isolates of both <i>A. baumannii</i> and <i>C. albicans</i> were co-isolated from urine samples with UTIs, then mono-cultured and co-cultured at 24, 48, and 72 h for biofilm quantification by crystal violet assay. The expression levels of bacterial (<i>ompA, bap, abaI</i>) and fungal (<i>ALS3, HWP1, ERG11</i>) virulence genes were evaluated by RT-qPCR at 24 and 48 h. Co-culture conditions resulted in increased biofilm biomass compared to monoculture for the tested isolates. In <i>A. baumannii,</i> the virulence genes of <i>bap, abaI, and ompA</i> showed statistically significant increase in expression in co-culture compared to mono-culture after 24 h. In <i>C. albicans</i>, <i>HWP1</i> is the only virulence gene that shows a statistically significant increase in expression in co-culture compared to monoculture after 48 h. Gene expression patterns varied in patient isolates, suggesting strain heterogeneity. This is an exploratory study that provides evidence of changes in biofilm formation and virulence gene expression in co-culture conditions among clinical isolates of <i>A. baumannii</i> and <i>C. albicans</i>. These findings suggest potential for microbial interactions under polymicrobial conditions, which might affect the diagnosis and treatment of patients with UTIs. Future studies with a larger number of isolates and functional assays are required to clarify the mechanistic regulation and biological relevance of these observations in UTIs.</p>