Glass fiber-based materials have gained interest for use in biomedical and dental applications. The aim of this study was to make E-glass fiber bioactive by a novel method using the microwave irradiation technique. Industrial E-glass fibers were used after surface activation with the hydrolysis method. The ratio of calcium and phosphorous precursors was set at 1.67. After maintaining the pH of the calcium solution, E-glass fibers in two ratios, i.e. 30% (nHA/E30) and 50% (nHA/E50) wt/wt, were added. The phosphorous precursor was added later and the solution was irradiated in a microwave to obtain nano-hydroxyapatite (nHA) particles on E-glass fibers. The structural, physical and in vitro biocompatibility analyses of the resulting materials were conducted. The expression of osteopontin (OPN) and collagen (Col) type 1 was measured by reverse transcription polymerase chain reaction (RT-PCR) and comparison was made between all the groups. Fourier transform infrared spectroscopy and x-ray diffraction showed characteristic peaks of nHA, and a change in the peak intensities was observed with an increase in the concentration of E-glass fibers. Scanning electron microscopic (SEM) images confirmed the homogenous adhesion of nHA spherical particles all over the fibers. Cell viability with mesenchymal stem cells showed growth, proliferation, and adhesion. All the materials were able to upregulate the expression of the OPN and Col, where gene expression was highest in nHA followed by nHA/E30 and nHA/E50. The bioactive glass fibers were synthesized in the shortest time and showed osteogenic properties. These materials have the potential for use in bone tissue engineering, dental prosthesis, and tooth restoration.