Oral microbial communities are in direct contact with tobacco smoking, which may affect these communities. This study aimed to investigate whether tobacco smoking alters the microbial diversity in oral fluid, with a focus on Gram-positive bacteria. Forty samples were collected from students at the Faculty of Technology at Sabha University, Libya during 2025. The participants were then divided into two groups: Group 1: Smokers and Group 2: Non-smokers. All the smokers sampled were found to be infected, while the non-smoker students did not have the infection or the type of bacteria required for the study. Two types of bacteria were found in the smokers: type 1 (Streptococcus) and type 2 (Staphylococcus). More people were infected with type 1 than with type 2. Among type 1 smokers, nine smokers did not suffer from dental caries, while five smokers had dental caries and were infected. These findings confirm that tobacco smoking significantly affects Gram-positive salivary microbes.