The medicinal value of the plants is due to their chemical components that bring a definite physiological action on the human body to prevent the diseases. In this work, we investigated the antimicrobial activity of leaves’ extracts of Quercus robur L., collected from the Algerian upper highlands, on ten bacterial strains and one fungal strain known to be pathogenic. First, we performed a qualitative phytochemical analysis, and second, antimicrobial activity tests performed by agar diffusion method (disc and well) with the determination of MIC by broth macro-dilution method. Given the results, it appears that obtained macerates of Quercus robur L. were rich in bioactive phytoconstituents such as alkaloids, anthraquinones, saponins, tannins, and other components. The yield of aqueous and methanolic macerates of leaves was 8.5 ± 1.41 and 22.4 ± 4.36%, respectively. The bacterial resistance was relatively important to several antibiotics, namely, ampicillin, amoxicillin + clavulanic acid for strains of Escherichia coli and Salmonella sp. However, Staphylococcus aureus strains were resistant to fusidic acid, penicillin, and oxacillin; while Enterococcus faecalis was resistant to fusidic acid, penicillin, oxacillin, and ticarcillin. The antibacterial activity of the macerates toward tested microbial strains showed that the aqueous and methanolic macerates of the leaves were proportional to the tested concentration and active not only against Gram-positive and Gram-negative bacteria but also on the fungal species Candida albicans. The estimated MIC for Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus was in the order of 10 mg/mL, which seems more effective than toward Salmonella sp., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans which were in the order of 30 mg/mL. These preliminary results confirm that the part of the studied plant had a very good antimicrobial activity that was proportional to the serial concentrations of the tested extracts.