The purpose of this study was to examine the changes in co-activation around the knee joint during different walking speeds in healthy females using the co-activation index. Ten healthy females (age: 21.20 ± 7.21 years, height: 164.00 ± 4.00 cm, mass: 60.60 ± 4.99 kg) participated in this study and performed three walking speeds (slow, normal, and fast). A Qualisys 11-camera motion analysis system sampling at a frequency of 200 Hz was synchronized with a Trigno EMG Wireless system operating at a 2000 Hz sampling frequency. A significant decrease in the co-activation index of thigh muscles was observed between the slow and fast, and between the normal and fast, walking speeds during all walking phases. A non-significant difference was observed between the slow and normal walking speeds during most walking phases, except the second double support phase, during which the difference was significant. A negative relationship was found between walking speed and the co-activation index of thigh muscles in all speeds during walking phases: first double support (r = −0.3386, p < 0.001), single support (r = −0.2144, p < 0.01), second double support (r = −0.4949, p < 0.001), and Swing (r = −0.1639, p < 0.05). In conclusion, the results indicated high variability of thigh muscle co-activation in healthy females during the different walking speeds, and a decrease in the co-activation of the thigh muscles with the increase of speed.