In this study, chitosan nanoparticles (CH-NPs) were synthesized using Penaeus semisulcatus shrimp shells and characterized using UV–Vis and FT-IR spectroscopy, as well as XRD and HR-TEM analyses. CH-NPs were investigated for growth inhibition properties against selected species of bacterial and fungal pathogens, showing performances higher or comparable over positive controls, respectively. Furthermore, CH-NPs were tested on three important mosquito vectors, achieving LC50 from 12.27 to 14.62 µg/ml. In addition, CH-NPs were evaluated using in vitro plant tissue culture by rooting gel method, to enhance the vegetative growth of the medicinal plant species Sphaeranthus indicus. With the simple technique presented here, large-scale industrial production of CH-NPs is possible. They can be used to develop pesticides highly effective against mosquito vectors of high medical and veterinary importance, as well as for plant tissue culture and food packaging applications.