The emergence of antibiotic-resistant bacteria has limited treatment options and led to the untreatable infections, thereby necessitating the discovery of new antibiotics to battel against bacteria. Natural products from endophytic actinobacteria (EA) serve as a reservoir for discovery of new antibiotics. Therefore, the current study focused on the isolation and antibacterial properties of EA isolated from Luffa cylindrica. Six strains were identified using morphological characterization, SEM analyses and 16S rRNA gene sequencing from the roots and leaves of the plant. They were taxonomically classified as Streptomycetaceae family. This is the first report on EA form L. cylindrica. The strains produced a chain of oval, cubed or cylindrical shaped spores with spiny or smooth surfaces. Three strains; KUMS-B3, KUMS-B4 and KUMS-B6 were reported as endophytes for the first time. Fifty percent of isolates were isolated from leaves samples using YECD medium. Our results showed that the sampling time and seasons may affect the bacterial diversity. All six strains had antibacterial activity against at least one of the tested bacteria S. aureus, P. aeruginosa, and E. coli. Among the strains, KUMS-B6 isolate, closely related to S. praecox, exhibited the highest antibacterial activity against both gram-positive and negative bacteria. KUMS-B6, KUMS-B5 and KUMS-B4 isolates strongly inhibited the growth of P. aeruginosa. Interestingly, the strains, isolated from leaves exhibited stronger antagonist activities compared to those isolated from the roots. The study revealed that the isolated strains from Luffa produce a plethora of bioactive substances that are potential source of new drug candidates for the treatment of infections.