ABSTRACT Objectives Oral biofilms initiate with the formation of an acquired pellicle on dental surfaces, a thin layer of salivary glycoproteins that provides a substrate for microbial adhesion. This study aimed to assess the necessity of a preformed pellicle for biofilm growth in vitro by analyzing the development of a standardized six‐species biofilm, comprising Actinomyces oris , Veillonella dispar , Fusobacterium nucleatum , Streptococcus sobrinus , Streptococcus oralis , and Candida albicans . Materials and Methods Biofilms were cultivated on bovine enamel discs under two conditions: (1) precoated with human saliva to simulate a pellicle and (2) without a preformed pellicle. Colony‐forming units (CFUs) of each microbial species were quantified after incubation in either human saliva or a NaCl‐based medium at 16 and 64 h. Results The analysis revealed no significant differences in CFU counts between discs with or without a preformed pellicle, regardless of whether biofilms were grown in human saliva or NaCl medium, with one exception: S. oralis in pellicle/NaCl (6.7 Log 10 ) medium at 16 h showed a slight decrease in the absence of a pellicle (5.9 Log 10 ). Conclusions These findings suggest that microbial adhesion and subsequent biofilm development occurred independently of an initial pellicle. The preformed salivary pellicle does not seem to play a significant role in the initial development of this in vitro biofilm model. Biofilm testing in laboratory settings, especially for studies on antimicrobial efficacy, could be simplified, as pellicle formation may not be an essential requirement. Although no significant differences in biofilm development were observed between pellicle and no‐pellicle conditions, the growth medium may have influenced pellicle interactions, warranting further investigation of media effects on pellicle formation. Existing assumptions about pellicle dependence in biofilm formation are challenged, and suggest that in vitro models without a pellicle may still provide valid platforms for studying biofilms and testing antimicrobial agents effectively.