Objectives: The goal of this investigation is to examine the ability of forming biofilm in clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa on two different broths - Meuller-Hinton and Luria-Bertani, as well as to compare the ability to form biofilm among the two mentioned types of bacteria and the ability of detecting the biofilm considering in vitro conditions, that is broths. Material and methods: 30 strains of Pseudomonas aeruginosa and 30 strains of Staphilococcus aureus obtained from clinical specimens were used for this investigation. After preparing the suspensions of bacteria inoculated on broths, they were set on microtiter plates and the biofilm production was measured using the spectrophotometric reader on 550 nm. The final results based on the formulas were classified into four categories. Results: The results showed that both of the tested strains, successfully and statistically significant, form a biofilm in both of the above mentioned broths. The results clearly indicate that for the gram negative P.aeruginosa strain in both broths the highest percentage of occurrence had a strong production, whereas in S.aureus a strong production appeared only in one measurement. Furthermore, there is no statistically significant difference in the biofilm formation between the strains on neither of the used strain and the same results occured after comparing the formation between the broths in both of the bacteria. Conclusion: The results of this investigation indirectly point to known cognitions of pathogenicity and virulence of these agents and can contribute to the better understanding of their in vivo characteristics in causing of infections related to biofilm.