In vitro propagation was developed for the medicinal plant Pereskia grandifolia Haworth var. grandifolia using axillary bud explants. Optimum multiplication of shoots was achieved on MS (Murashige and Skoog, 1962) medium supplemented with 3.0 mg/l BAP (6-Benzylaminopurine) and 30.0 mg/l adenine. Plants were maintained in vitro on MS medium while callus were induced on MS basal medium supplemented with the combination of 5.0 mg/l BAP (6-Benzylaminopurine) and 5.0 mg/l NAA (α- Naphthalene acetic acid). The antimicrobial tests were carried out using the crude extracts of this species from in vitro callus and intact leaves. No difference was detected between crude methanol extract from Pereskia grandifolia leaves and callus. The same extract failed to inhibit all gram positive and gram negative bacteria growth; however, it inhibited the growth of fungi. Only dermatophytes fungi (without spore) such as Microsporium canis and Trichophyton rubrum was inhibited with a medium and weak strength of methanol extract. This extract did not inhibit the growth of dermatophytes fungi (with spore) such as Aspergillus niger and Trichophyton mentagrophyte.