Citrate ion is a commonly used reductant in metal colloid synthesis, undergoes strong surface interaction with silver nanocrystallites. The slow crystal growth observed as a result of the interaction between the silver surface and the citrate ion makes this reduction process unique compared to other chemical and radiolytic synthetic methods. The antimicrobial effects of silver (Ag) ion or salts are well known, but the effects of citrate coated Ag nanoparticles (CAgNPs) are scant. Herein, we have isolated biofilm causative bacteria and fungi from drinking water PVC pipe lines. Stable CAgNPs were prepared and the formation of CAgNPs was confirmed by UV-visible spectroscopic analysis and recorded the localized surface plasmon resonance of CAgNPs at 430 nm. Fourier transform infrared spectroscopic analysis revealed C=O and O-H bending vibrations due to organic capping of silver responsible for the reduction and stabilization of the CAgNPs. X-ray diffraction micrograph indicated the face centered cubic structure of the formed CAgNPs, and morphological studies including size (average size 50 nm) were carried out using transmission electron microscopy. The hydrodynamic diameter (60.7 nm) and zeta potential (-27.6 mV) were measured using the dynamic light scattering technique. The antimicrobial activity of CAgNPs was evaluated (in vitro) against the isolated fungi, Gram-negative and Gram-positive bacteria using disc diffusion method and results revealed that CAgNPs with 170ppm concentration are having significant antimicrobial effects against an array of microbes tested.