Natural products are sources of inspiration and reservoir of high valuable molecules. Recently, analytical tools based on liquid chromatography coupled to tandem mass spectrometry to generate molecular network became widely employed for dereplication. This strategy greatly accelerates the identification of known and structural hypothesis of unknown. Despite the availability of different ionization sources, alternatives to classical electrospray ionization (ESI), such as atmospheric pressure chemical ionization (APCI) or photoionization (APPI), have been neglected. In particular, APPI has been described for its ionization efficiency on non-polar molecules bearing no acid or basic groups. For that reason, we investigated APPI potential to generate molecular network and compare it to ESI on several criteria that are generation of ion species, sensitivity and signal-to-noise ratio (SNR) for different extracts rich in highly conjugated natural products. We first optimized APPI experimental conditions on crude extract from a fungus, Penicillium sclerotiorum, producing polyketones belonging to the azaphilone family. Then we compared APPI and ESI on different fractions of the fungus and on two plant extracts, French Guyanese Swartzia panacoco (Aubl.) R.S. Cowan (arial parts) and Indian Cassia auriculata L. (leaves) containing phenolic compounds, such as flavonoids. While ESI generated more ion species and displayed a better sensitivity, APPI generated only protonated adduct and better SNR. Comparing ESI and APPI generated species on molecular network reveal that both strategies overlap for the majority of protonated ions.