In the current study, we investigated the mechanism of medium-scale traveling ionospheric disturbance (MSTID) triggering spread-F in the low latitude ionosphere using ionosonde observation and Global Navigation Satellite System-Total Electron Content (GNSS-TEC) measurement. We use a series of morphological processing techniques applied to ionograms to retrieve the O-wave traces automatically. The maximum entropy method (MEM) was also utilized to obtain the propagation parameters of MSTID. Although it is widely acknowledged that MSTID is normally accompanied by polarization electric fields which can trigger Rayleigh–Taylor (RT) instability and consequently excite spread-F, our statistical analysis of 13 months of MSTID and spread-F occurrence showed that there is an inverse seasonal occurrence rate between MSTID and spread-F. Thus, we assert that only MSTID with certain properties can trigger spread-F occurrence. We also note that the MSTID at night has a high possibility to trigger spread-F. We assume that this tendency is consistent with the fact that the polarization electric field caused by MSTID is generally the main source of post-midnight F-layer instability. Moreover, after thorough investigation over the azimuth, phase speed, main frequency, and wave number over the South America region, we found that the spread-F has a tendency to be triggered by nighttime MSTID, which is generally characterized by larger ΔTEC amplitudes.