Due to the significance of silver nanoparticles (AgNPs) in the environmental area and their distinctive physicochemical characteristics, the use of plant extracts for their production has garnered attention from all over the world. In this study, Kalanchoe tubiflora (K. tubiflora) leaf extract was used for the synthesis of Kt-AgNPs. A series of techniques such as UV–visible spectroscopy, FTIR, XRD, HRTEM, SEM, and EDS analysis were used to characterise the synthesized nanoparticles. Additionally, the antibacterial activity, multimode antioxidant potentials, cytotoxic activities, and protein kinase inhibition were established. The intense UV–Visible peak was observed at 444 nm for the fabricated Kt-AgNPs. Also the FTIR spectra, afforded the bands for proteins N–H vibrations and carboxylic acids C = O functional groups that are accountable for the fabricated AgNPs stability. Moreover, the 111 XRD peak confirmed the crystalline sizes (CS) of the resulting AgNPs between 10 nm and 17 nm. Moreover, in the antibacterial activity the nanoparticles exhibited the highest activity against B. bronchiseptica (P < 0.05) with a growth inhibition zone of 18 ± 1.75 mm compared to the plant extract (11 ± 1.23 mm). Also, Kt-AgNPs showed the highest DPPH scavenging activity (88%), antioxidant capacity (115.12 ± 1.11 µgAAE/mg), and anti-reducing power (80.53 ± 0.77 µgAAE/mg) activities compared to the extract. In conclusion, in this study for the first time the Kt-AgNPs were prepared using K. tubiflora extract by optimizing the conditions regarding the AgNO3: extract ratio, pH, temperature etc. That might lessen the difficulties currently facing Kt-AgNPs growth in the disciplines of biotechnology and chemical engineering, and it might also indicate where Kt-AgNPs might go in the future.