The isolation of nanocellulose from lignocellulosic biomass, with desirable surface chemistry and morphology, has gained extensive scientific attention for various applications including polymer nanocomposite reinforcement. Additionally, environmental and economic concerns have driven researchers to explore viable alternatives to current isolation approaches, employing chemicals with reduced environmental impact. To address these issues, in this study, we have tuned the amphiphilic behavior of cellulose nanofibers (CNFs) by employing controlled alkali treatment, instead of in combination with expensive, environmentally unsustainable conventional approaches. Microscopic and spectroscopic analysis demonstrated that this approach is capable of tuning composition and interfacial tension of CNFs through a careful control of the quantity of residual lignin and hemicellulose. To elucidate the performance of CNF as an efficient reinforcing nanofiller in hydrophobic polymer matrices, prevulcanized natural rubber (NR) latex was employed as a suitable host polymer. CNF/NR nanocomposites with different CNF loading levels (0.1–1 wt % CNF) were prepared by a casting method. It was found that the incorporation of 0.1 wt % CNF treated with a 0.5 w/v % sodium hydroxide solution led to the highest latex reinforcement efficiency, with an enhancement in tensile stress and toughness of 16% to 42 MPa and 9% to 197 MJ m–3, respectively. This property profile offers a potential application for the high-performance medical devices such as condoms and gloves.