This paper investigates micromachined antenna performance operating at 5 GHz for radio frequency (RF) energy harvesting applications by comparing different substrate materials and fabrication modes. The research aims to discover appropriate antenna designs that can be integrated with the rectifier circuit and fabricated in a CMOS (Complementary Metal-Oxide Semiconductor)-compatible process approach. Therefore, the investigation involves the comparison of three different micromachined antenna substrate materials, including micromachined Si surface, micromachined Si bulk with air gaps, and micromachined glass-surface antenna, as well as conventional RT/Duroid-5880 (Rogers Corp., Chandler, AZ, USA)-based antenna as the reference. The characteristics of the antennas have been analysed using CST-MWS (CST MICROWAVE STUDIO®-High Frequency EM Simulation Tool). The results show that the Si-surface micromachined antenna does not meet the parameter requirement for RF antenna specification. However, by creating an air gap on the Si substrate using a micro-electromechanical system (MEMS) process, the antenna performance could be improved. On the other hand, the glass-based antenna presents a good S11 parameter, wide bandwidth, VSWR (Voltage Standing Wave Ratio) ≤ 2, omnidirectional radiation pattern and acceptable maximum gain of >5 dB. The measurement results on the fabricated glass-based antenna show good agreement with the simulation results. The study on the alternative antenna substrates and structures is especially useful for the development of integrated patch antennas for RF energy harvesting systems.