In this study, 63 isolates of actinobacteria were isolated from marine sediment and sandy soil of Saint Katherine, Egypt and examined for antimicrobial activity. Both sites were highly inhabited by rare promising Actinobacteria species. Recovered Actinobacteria belonging to genus Streptomyces, Actinomadura, Micromonospora, Microtetraspora, Nocardia, Pseudonocardia Nocardiopsis, Actinoplanes, Pilimelia and Spirellospora. Their extracts exhibited wide antimicrobial activities towards 4 clinical pathogens. Out of them 49.21% showed potent inhibitory effect against test pathogens in primary and secondary screening methods in which genus Actinomadura was significantly the highest followed by genus Streptomyces then genus Micromonospora. The crude extracts showed 0.3-20 mm inhibition zone in disc diffusion method against tested pathogens. The active compounds were purified by preparative TLC, which showed retention factor value (RF) of 0.14-0.857 with isolated bioactive Actinobacteria and identified using different reference antibiotics. In bioautography, TLC spot of Actinomadura roseoviolacea with RF of 0.26 showed maximum activity with 40 mm inhibition zone and nearby Tetracycline reference antibiotic. This isolate show safe hemolytic activity In vitro analysis.