Kenyans have long utilized Ocimum kilimandscharicum, an East African permanent evergreen plant, to treat measles, stomachaches, diarrhea, mosquito bites (anti-insect), congested chest, cough, and colds. Using conventional qualitative and quantitative techniques, this study was done to identify the secondary metabolites in O. kilimandscharicum leaf extracts. The chemical content of the crude extracts from the leaves of O. kilimandscharicum has also been investigated and characterized using gas chromatography-mass spectrometry (GC-MS). By using a 1:20 dilution in methanol, in cold maceration, a fine powder of O. kilimandscharicum was first extracted then filtered and concentrated after 72 h utilizing a rotary evaporator. By using also a 1:20 dilution in water at 80 °C, a fine powder of O. kilimandscharicum was extracted and then filtrated and lyophilized 1 h later. Each extract underwent further gas chromatography-mass spectrometry testing. We found that both extracts contain secondary metabolites such as alkaloids, phenolics, flavonoids, saponins, and tannins. However, the overall amount of phytochemicals in each solvent varied significantly. Total phenolics contents (TPCs) were 5.6 ± 1.20 and 10.8 ± 1.00 mg, total flavonoid contents (TFCs) were 8.2 ± 0.4 and 39.6 ± 2.2 mg, total tannin contents (TTCs) were 0 ± 0.00 and 10.5 ± 0.4 mg, the total alkaloid content (TAC) was 49.2 ± 0.40%, and the total saponin content (TSC) was 38 ± 2.00%. Additionally the gas chromatography-mass spectrometry, revealed a number of high- and low-molecular-weight bioactive molecules at various concentrations for each extract. We also found an inhibitory effect on adhP and chbR gene expression of Staphylococcus aureus and Salmonella choleraesuius, respectively. Hence, these chemicals could potentially have a biological and pharmacological significance. Therefore, the discovery of many physiologically active chemicals in the leaf extracts of O. kilimandscharicum justifies future biological and pharmaceutical research.