Aims: The focus of this study was to isolate and to identify strains with antibacterial activity followed by a partial characterization of their extracts. Study Design: Screening and identification of bacteria having an anti-mycobacterial effect from soil and water of different biotopes of Fez Morocco were performed and active substances responsible for the biological activity were partially characterized. Place and Duration of Study: The study was carried out at laboratory of Microbial Biotechnology, Department of Biology, Faculty of Sciences and Technical, University Sidi Mohamed Ben Abdellah, BP 2202, Road of Immouzer, Fez, Morocco, during the period from January 2011 to October 2011. Methodology: Samples of soil and water of different biotopes of Fez Morocco were explored to isolate... compounds-producing microorganisms. The inhibitory spectrum of the isolated bacteria was evaluated against M. smegmatis, M. aurum, S. aureus, S. haemolyticus, B. subtilis, E. coli DH5α, P. aeruginosa and Erwinia chrysanthemi by using agar well diffusion test and/or a modified spot-on-lawn assay. Identification of strains was executed on the basis of Gram stain, biochemical characteristics and PCR followed by DNA sequencing of 16S ribosomal RNA gene. Crude extracts obtained after precipitation by ammonium sulfate were exposed to proteolytic enzymes (Pepsin and proteinase K) and to heat treatment at 100ºC (15 min), 80°C (30 min), 37ºC (3h) and 4ºC (1 month). The residual activity after every treatment was assessed by agar well diffusion method. Results: Six bacteria were isolated from different biotopes of Fez Morocco having an antibacterial effect against M. smegmatis, M. aurum, Gram-positive and Gram-negative bacteria. Based on biochemical characterization and 16S rDNA sequence analysis, it was revealed that the isolates belong to the genus Bacillus. The antibacterial activity of three of them was fully affected by proteases and heat treatment at 80ºC and 100ºC but it was stable at 4ºC for a month and 37ºC during 3h. Conclusion: The lost of the activity suggests a proteinaceous nature of the bio-active compounds, which might be useful in the development of antibacterial agents after their total purification in further work against bacterial infections.