Bacterial vaginosis (BV) is characterized by a highly structured polymicrobial biofilm, which is strongly adhered to the vaginal epithelium and primarily consists of the bacterium Gardnerella vaginalis. However, despite the presence of other BV-associated bacteria, little is known regarding the impact of other species on BV development. To gain insight into BV progress, we analyzed the ecological interactions between G. vaginalis and 15 BV-associated microorganisms using a dual-species biofilm model. Bacterial populations were quantified using a validated peptide nucleic acid fluorescence in situ hybridization approach. Furthermore, biofilm structure was analyzed by confocal laser scanning microscopy. In addition, bacterial coaggregation ability was determined as well as the expression of key virulence genes. Remarkably, our results revealed distinct biofilm structures between each bacterial consortium, leading to at least three unique dual-species biofilm morphotypes. Furthermore, our transcriptomic findings seem to indicate that Enterococcus faecalis and Actinomyces neuii had a higher impact on the enhancement of G. vaginalis virulence, while the other tested species had a lower or no impact on G. vaginalis virulence. This study casts a new light on how BV-associated species can modulate the virulence aspects of G. vaginalis, contributing to a better understanding of the development of BV-associated biofilms.