The Egyptian climate possesses many herbal medicinal plants, three species namely carnation (Dianthus caryophyllus), thyme (Thymus vulgaris L) and basil (Ocimum basilicumL) were selected to study in this research. The current study was designed to optimize a protocol of in vitro propagation for progeny uniformity mass production of the selected species to avoid their genetic and biochemical heterogeneity. Nodal segments of carnation, thyme and basil seedlings cultured on medium contained benzyl amino purine (BAP) at different concentrations with or without naphthalene acetic acid (NAA) at 0.5 mg/l. The presence of NAA combined with a high level of BAP was more efficient for shootlets mass production of all studied plant species. Shootlets were extracted using diethyl ether, most of their substantial phytocomponents (fatty acid derivatives, essential oils, , cyclohexane carboxylic acid, hydrocarbons, etc.,) were detected in different proportions by GC-MS analysis. Shootlets extracts were evaluated antimicrobial in vitro, and exhibited differentially good performance against Bacillus subtilis, Staphylococcus aureus, Pseudomonas floureceans, Escherichia coli and Candida albicans. Diameters of inhibition zones ranged from 16.133±0.696 to 23.466±0.837mm for all extracts. In conclusion, in vitro normal healthy shootlets of carnation, thyme and basil were obtained, and their extracts were rich in bioactive phytochemicals which in turns play a potent role for microbial growth inhibition.