Recent evidences have shown that methicillin-resistant Staphylococcus aureus (MRSA) can cause severe infections and is resistant to almost all commercially available antibiotics. Therefore, screening unknown sources of biological compounds such as the Thermoactinomycetaceae family as extremophilic bacteria may be helpful to find new antimicrobial agents. Various samples were collected from different ecosystems, including desert, volcano, compost, and forest. They were cultured on Soil extract agar and Water agar. The antimicrobial activity of the isolates was evaluated using agar overlay and well diffusion methods. Members of the Thermoactinomycetaceae family were selected for further study: Their ability to grow at different temperatures, NaCl concentrations, and pH values, enzyme production ability, antimicrobial secondary screening, fractionation of their supernatants and so on. According to molecular identification of active isolates against MRSA, three strains, including Laceyella sacchari UTMC 2705, Thermoactinomyces sp. UTMC 2721, and Laceyella sp. UTMC 2731, belonged to Thermoactinomycetaceae were identified. The minimum inhibitory concentrations of their extracts were tested against some pathogenic bacteria, showing their antimicrobial activity with a broad spectrum. The results of TLC bioautography of the extracts showed that the most active fractions were semi-polar. Also, the results of HPLC analysis showed the existence of several UV-active compounds in their extracts. The present study highlighted the importance and potential of Thermoactinomycetaceae members as a less-known source of antibiotics against pathogenic bacteria.