There is grave necessity to counter the menace of drug-resistant biofilms of pathogens using nanomaterials. Moreover, we need to produce nanoparticles (NPs) using inexpensive clean biological approaches that demonstrate broad-spectrum inhibition of microbial biofilms and cytotoxicity against HepG2 cell lines. In the current research work, titanium dioxide (TiO
2
) NPs were fabricated through an environmentally friendly green process using the root extract of
Withania somnifera
as the stabilizing and reducing agent to examine its antibiofilm and anticancer potential. Further, X-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), transmission electron micrograph (TEM), energy-dispersive X-ray spectroscopy (EDS), dynamic light scattering (DLS), thermogravimetric analysis (TGA), and Brunauer-Emmett-Teller (BET) techniques were used for determining the crystallinity, functional groups involved, shape, size, thermal behavior, surface area, and porosity measurement, respectively, of the synthesized TiO
2
NPs. Antimicrobial potential of the TiO
2
NPs was determined by evaluating the minimum inhibitory concentration (MIC) against
Escherichia coli
,
Pseudomonas aeruginosa
, methicillin-resistant
Staphylococcus aureus
,
Listeria monocytogenes
,
Serratia marcescens
, and
Candida albicans
. Furthermore, at levels below the MIC (0.5 × MIC), TiO
2
NPs demonstrated significant inhibition of biofilm formation (43–71%) and mature biofilms (24–64%) in all test pathogens. Cell death due to enhanced reactive oxygen species (ROS) production could be responsible for the impaired biofilm production in TiO
2
NP–treated pathogens. The synthesized NPs induced considerable reduction in the viability of HepG2
in vitro
and could prove effective in controlling liver cancer. In summary, the green synthesized TiO
2
NPs demonstrate multifarious biological properties and could be used as an anti-infective agent to treat biofilm-based infections and cancer.