Objective: To synthesize silver nanoparticles (AgNPs) by green methods using serum latex of Calotropis procera at 80 °C and evaluate them against bacteria, dermatophytes and phytopathogenic fungi comparing with the activity of untreated latex. Methods: The synthesis of AgNPs was performed by mixing 3% latex serum extract with the same volume of silver nitrate (2 mmol/L) solution in round flask and heating in water bath at 80°C. Characterization of silver particles were determined using UV-vis spectrophotometer, transmission electron microscopy (TEM), X-ray diffraction and Fourier transform infrared spectroscopy. The antimicrobial activity of the green synthesized AgNPs was determined against bacteria, dermatophytes and phytopathogenic fungi and compared to the crude untreated latex by agar-well diffusion methods. Results: Biosynthesis of latex silver nanoparticles was successfully obtained by green method. The formation of AgNPs has been confirmed by UV-vis, TEM microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. TEM analysis showed that synthesized AgNPs are highly stable spherical shaped particles, well dispersed with a diameter ranged from 4 nm up to 25 nm and an average size of 12.33 nm. AgNPs showed strong antibacterial activity against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Serratia sp.) and antifungal activity against Trichophyton rubrum, Candida albicans and Aspergillus terreus. Conclusions: It can be concluded that serum latex of Calotropis procera was found to display strong potential for the synthesis of AgNPs as antimicrobial agents through rapid reduction of silver ions (Ag