Due to the various intensities of 5-aminolevulinic acid (5-ALA) fluorescence, neurosurgeons tend to be uncertain about which tissues to resect. This study aimed to reveal the shortcomings of the human visual perception of fluorescence, particularly the factors guiding the tissue removal and the correlation of fluorescence with contrast enhancement (CE) on magnetic resonance imaging (MRI). Various colour features [CIE L*a*b* colour space, colour difference described by ΔE and contrast ratio (CR)] of total 206 noticed fluorescent areas and their surroundings were measured from the video recordings of 21 primary high grade glioma (HGG) surgeries. The position of a fluorescent region was related to the corecorded navigational image. Following early postoperative MRI, 17 additional regions of corresponding to CE remnants were identified, their colour features were compared to the resected CEs. The targeted video post-processing method was designed, based on the results. There were no complications attributed to 5-ALA use and the median survival was <10 months. 82.5 % of recognised fluorescent areas were removed. Colour spaces of the resected regions and their backgrounds did not overlap. Opposite to the separate colour components (p > 0.05), the distant background colour (p < 0.05) and higher CR and ΔE (p < 0.01) determined the resection of a fluorescent region. Noneloquent location and CR both independently increased the resection rate in logistic regression. However, greater area under the receiver operating characteristic curve (AUC) in case of CR (AUC = 0.78; 95 % CI 0.71-0.83) determined its dominant role in neurosurgeon's fluorescence perception. CE regions presented with a significantly more saturated shade of violet (consistently higher a* and b*) than other tumour parts (p < 0.05). Regions corresponding to tumour remnants had a significantly lower a* component value (p = 0.02) as well as a lower ΔE than the matched background (AUC = 0.73; 95 % CI 0.65-0.80). In order to increase the resection rate, ΔE > 60 was needed. These results directed essential improvements in the 5-ALA fluorescence visualisation toward enhanced resection rate. The conventional filtering, unadjusted to the 5-ALA colour space converted some background shades to colours resembling relevant fluorescence. This is one of the first studies to demonstrate that perceived colours, their contrasting and CR are of significance in the decision-making during HGG 5-ALA fluorescence-guided surgery. Irrespective of the shortcomings of conventional video filtering, further development of a tailored post-processed contrast stretching will allow to achieve safe and radical tumour resection.