The phytochemical study and in vitro antimicrobial studies of further fractionation of the most active column fraction of the ethanolic extract of stem-bark of Zizyphusspina christi Linn were evaluated using Gram positive and Gram negative pathogens as well as Candidaalbican using the disc diffusion method. The powdered most active column fraction (15.8g) was further fractionated using hexane / chloroform methanol as eluting solvents. The resulting portions from column chromatographic separation were combined into five (5) fractions A1–A5 on the basis of TLC results. However fraction A5 was pure compound obtained after crystallization using acetone.Fractions A1to A4 were again recombined based on polarity where (A1& A2) were of lower polaritythan (A3& A4). Phytochemical analysis showed moderate presence of terpenoids and cardiac glycoside in (A1& A2), while fraction (A3& A4) showed copious presence of cardiac glycoside, moderate presence of terpenoids and alkaloids but low flavonoid content. The pure compound showed low presence of terpenoids and cardiac glycosides. Anti-microbial susceptibility studies showed that column fractions (A1& A2) and (A3& A4) were very active against all organisms tested except Escherichia coli,Streptococcus pyogenes and Pseudomonas aeruginosa while the pure compound was active only on Proteus mirabilis and Bacillus subtilis. The pure compound after spectroscopic studies was confirmed to be betulinic acid. The MIC values of the susceptible organism ranged from 6.25 – 18.75, while the MBC were usually one step higher than the MIC (12.25 to 25). The reference antibiotic showed the same pattern in the MIC and MBC. The fact that the MBC values were only one step away from the MIC implies that the activity of the column fractions were bactericidal. Since betulinic acid, a triperpenoidhas antimicrobial activity; the compound maybe partlyresponsible for the antimicrobial activity of Z. spina-christi. The results have provided scientific validity for the use of this plant in the treatment of bacteria-related infections in herbal medicines.