Agro-industrial wastes are rich in polyphenols and other bioactive compounds, and valorizing these wastes is a crucial worldwide concern for saving health and the environment. In this work, olive leaf waste was valorized by silver nitrate to produce silver nanoparticles (OLAgNPs), which exhibited various biological, antioxidant, anticancer activities against three cancer cell lines, and antimicrobial activity against multi-drug resistant (MDR) bacteria and fungi. The obtained OLAgNPs were spherical, with an average size of 28 nm, negatively charged at −21 mV, and surrounded by various active groups more than the parent extract based on FTIR spectra. The total phenolic and total flavonoid contents significantly increased in OLAgNPs by 42 and 50% over the olive leaf waste extract (OLWE); consequently, the antioxidant activity of OLAgNPs increased by 12% over OLWE, recording an SC50 of OLAgNPs of 5 µg/mL compared to 30 µg/mL in the extract. The phenolic compound profile detected by HPLC showed that gallic acid, chlorogenic acid, rutin, naringenin, catechin, and propyl gallate were the main compounds in the HPLC profile of OLAgNPs and OLWE; the content of these compounds was higher in OLAgNPs than OLWE by 16-fold. The higher phenolic compounds in OLAgNPs are attributable to the significant increase in biological activities of OLAgNPs than that of OLWE. OLAgNPs successfully inhibited the proliferation of three cancer cell lines, MCF-7, HeLa, and HT-29, by 79–82% compared to 55–67% in OLWE and 75–79% in doxorubicin (DOX). The preliminary worldwide problem is multi-drug resistant microorganisms (MDR) because of the random use of antibiotics. Therefore, in this study, we may find the solution in OLAgNPs with concentrations of 2.5–20 µg/mL, which significantly inhibited the growth of six MDR bacteria L. monocytogenes, B. cereus, S. aureus, Y. enterocolitica, C. jejuni, and E. coli with inhibition zone diameters of 25–37 mm and six pathogenic fungi in the range of 26–35 mm compared to antibiotics. OLAgNPs in this study may be applied safely in new medicine to mitigate free radicals, cancer, and MDR pathogens.